DNA immunization against Chlamydia infection

ABSTRACT

Nucleic acid, including DNA, for immunization to generate a protective immune response in a host, including humans, to a major outer membrane protein of a strain of  Chlamydia , preferably contains a nucleotide sequence encoding a MOMP or a MOMP fragment that generates antibodies that specifically react with MOMP and a promoter sequence operatively coupled to the first nucleotide sequence for expression of the MOMP in the host. The non-replicating vector may be formulated with a pharmaceutically-acceptable carrier for in vivo administration to the host.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of copending U.S.application Ser. No. 10/036,507 filed Jan. 7, 2002 (now U.S. Pat. No.6,838,085), which itself is a continuation-in-part of U.S. patentapplication Ser. No. 08/893.381 filed Jul. 11, 1997 (now U.S. Pat. No.6,235,290), which claims priority pursuant to 35 USC 119(e) from USProvisional Patent Application No. 60/021,607 filed Jul. 12, 1996.

FIELD OF INVENTION

The present invention relates to immunology and, in particular, toimmunization of hosts using nucleic acid to provide protection againstinfection by Chlaymdia.

BACKGROUND OF THE INVENTION

DNA immunization is an approach for generating protective immunityagainst infectious diseases (ref. 1 —throughout this application,various references are cited in parentheses to describe more fully thestate of the art to which this invention pertains. Full bibliographicinformation for each citation is found at the end of the specification,immediately preceding the claims. The disclosure of these references arehereby incorporated by reference into the present disclosure). Unlikeprotein or peptide based subunit vaccines, DNA immunization providesprotective immunity through expression of foreign proteins by hostcells, thus allowing the presentation of antigen to the immune system ina manner more analogous to that which occurs during infection withviruses or intracellular pathogens (ref. 2). Although considerableinterest has been generated by this technique, successful immunity hasbeen most consistently induced by DNA immunization for viral diseases(ref. 3). Results have been more variable with non-viral pathogens whichmay reflect differences in the nature of the pathogens, in theimmunizing antigens chosen, and in the routes of immunization (ref. 4).Further development of DNA vaccination will depend on elucidating theunderlying immunological mechanisms and broadening its application toother infectious diseases for which existing strategies of vaccinedevelopment have failed.

Chlamydia trachomatis is an obligate intracellular bacterial pathogenwhich usually remains localized to mucosal epithelial surfaces of thehuman host. Chlamydia are dimorphic bacteria with an extracellularspore-like transmission cell termed the elementary body (EB) and anintracellular replicative cell termed the reticulate body (ref. 5). Froma public health perspective, chlamydial infections are of greatimportance because they are significant causes of infertility, blindnessand are a prevalent co-factor facilitating the transmission of humanimmunodeficiency virus type 1 (ref. 6). Protective immunity to C.trachomatis is effected through cytokines released by Th1-like CD 4lymphocyte responses and by local antibody in mucosal secretions and isbelieved to be primarily directed to the major outer membrane protein(MOMP), which is quantitatively the dominant surface protein on thechlamydial bacterial cell and has a molecular mass of about 40 kDa (ref.19).

Initial efforts in developing a chlamydial vaccine were based onparenteral immunization with the whole bacterial cell. Although thisapproach met with success in human trials, it was limited becauseprotection was short-lived, partial and vaccination may exacerbatedisease during subsequent infection episodes possibly due topathological reactions to certain chlamydial antigens (ref. 8). Morerecent attempts at chlamydial vaccine design have been based on asubunit design using MOMP protein or peptides. These subunit vaccineshave also generally failed, perhaps because the immunogens do not induceprotective cellular and humoral immune responses recalled by nativeepitopes on the organism (ref. 9).

EP 192033 describes the provision of DNA construct for the expression,in vitro, of Chlamydia trachomatis MOMP polypeptides comprising thefollowing operably linked elements:

a transcriptional promoter,

a DNA molecule encoding a C. trachomatis MOMP polypeptide comprising aMOMP polynucleotide at least 27 base pairs in length from a sequenceprovided in Appendix A thereto, and

a transcriptional terminator, wherein at least one of thetranscriptional regulatory elements is not derived from Chlamydiatrachomatis. There is no disclosure or suggestion in this prior art toeffect DNA immunization with any such constructs.

WO 94/26900 describes the provision of hybrid picornaviruses whichexpress chlamydial epitopes from MOMP of Chlamydia trachomatis and whichis capable of inducing antibodies immuno-reactive with at least threedifferent Chlamydia serovars. The hybrid picornavirus preferably is ahybrid polio virus which is attenuated for human administration.

SUMMARY OF THE INVENTION

The present invention is concerned with nucleic acid immunization,specifically DNA immunization, to generate in a host protectiveantibodies to a MOMP of a strain of Chlamydia. DNA immunization inducesa broad spectrum of immune responses including Th1-like CD4 responsesand mucosal immunity.

Accordingly, in one aspect, the present invention provides animmunogenic composition for in vivo administration to a host for thegeneration in the host of a protective immune response to a major outermembrane protein (MOMP) of a strain of Chlamydia, comprising anon-replicating vector comprising a nucleotide sequence encoding a MOMPor MOMP fragment that generates a MOMP-specific immune response, and apromoter sequence operatively coupled to the nucleotide sequence forexpression of the MOMP or MOMP fragment in the host; and apharmaceutically-acceptable carrier therefor.

The nucleotide sequence may encode a full-length MOMP protein or mayencode a fragment, such as the N-terminal half of MOMP or a fragmentthat encompasses epitopic sequences. The nucleotide sequence may encodea MOMP or MOMP fragment which stimulates a recall immune responsefollowing exposure to wild-type Chlamydia. The promoter may be thecytomegalovirus promoter.

The fragment that encompasses epitopic sequences may include one or moreconserved domain (CD) sequences and/or one or more variable domain (VD)sequences of MOMP from a strain of Chlamydia. In particular, thefragment may encompass the CD2 and VD2 sequences, CD3 and VD3 sequencesand CD5 sequence. Clones containing nucleotide sequences encoding suchfragments are termed clones CV2, CV3 and CD5 herein. Clones CV2encompasses nucleotides 247 to 468 of Chlamydia trachomatis MOMP gene,clone CV3 encompasses nucleotides 469 to 696 of Chlamydia trachomatisMOMP gene and clone CV5 encompasses nucleotides 931 to 1098 of Chlamydiatrachomatis MOMP gene. Non-replicating vectors comprising such sequencesare novel and constitute further aspects of the invention.

Accordingly, in an additional aspect of the invention, there is provideda non-replicating vector, comprising a nucleotide sequence encoding aregion comprising at least one of the conserved domains 2, 3 and 5 of amajor outer membrane protein of a strain of Chlamydia, and a promotersequence operatively coupled to the nucleotide sequence for expressionof the at least one conserved domain in a host. In this aspect of theinvention, the various options and alternatives discussed above andbelow may be employed.

The strain of Chlamydia may be a strain of Chlamydia inducing chlamydialinfection of the lung, including Chlamydia trachomatis or Chlamydiapneumoniae. The non-replicating vector may be plasmid pcDNA3 into whichthe nucleotide sequence is inserted. The immune response which isstimulated may be predominantly a cellular immune response.

In a further aspect of the invention, there is provided as a method ofimmunizing a host against disease caused by infection with a strain ofChlamydia, which comprises administering to the host an effective amountof a non-replicating vector comprising a nucleotide sequence encoding amajor outer membrane protein (MOMP) of a strain of Chlamydia or a MOMPfragment that generates a MOMP-specific immune response, and a promotersequence operatively coupled to the nucleotide sequence for expressionof the MOMP or MOMP fragment in the host.

In this aspect of the present invention, the various options andalternatives discussed above may be employed.

The non-replicating vector may be administrated to the host, including ahuman host, in any convenient manner, such as intramuscularly orintranasally. Intranasal administration stimulated the strongest immuneresponse in experiments conducted herein.

The present invention also includes, in an additional aspect thereof, amethod of using a gene encoding a major outer membrane protein (MOMP) ofa strain of Chlamydia or MOMP fragment that generates a MOMP-specificimmune response, to produce an immune response in a host, whichcomprises isolating the gene, operatively linking the gene to at leastone control sequence to produce a non-replicating vector, the controlsequence directing expression of the MOMP or MOMP fragment whenintroduced into a host to produce an immune response to the MOMP or MOMPfragment, and introducing the vector into a host.

A further aspect of the present invention provides a method of producinga vaccine for protection of a host against disease caused by infectionwith a strain of Chlamydia, which comprises isolating a nucleotidesequence encoding a major outer membrane protein (MOMP) of a strain ofChlamydia or a MOMP fragment that generates a MOMP-specific immuneresponse, operatively linking the nucleotide sequence to at least onecontrol sequence to produce a non-replicating vector, the controlsequence directing expression of the MOMP or MOMP fragment whenintroduced to a host to produce an immune response to the MOMP or MOMPfragment, and formulating the vector as a vaccine for in vivoadministration to a host. The invention extends to the vaccine producedby this method.

Advantages of the present invention, therefore, include a method ofobtaining a protective immune response to infection carried by a strainof Chlamydia by nucleic acid immunization of nucleic acid sequenceencoding the major outer membrane protein of a strain of Chlamydia or afragment of the outer membrane protein that generates a MOMP-specificimmune response.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates delayed-type hypersensitively (DTH) responses in micefollowing immunization. Balb/c mice (four per group) were immunizedintramuscularly (pMOMP IM) or intranasally (pMOMP IN) with plasmid DNAcontaining the coding sequence of the MoPn MOMP gene or with MoPnelementary bodies (EB) at 0, 3, 6 weeks. The control group was treatedwith the blank plasmid vector (pcDNA3). Fifteen days after the lastimmunization, mice were tested for MoPn-specific DTH response asfollows: 25 μl of heat-inactivated MoPn EB (5×10⁴ IFU) in SPG buffer wasinjected into the right hind footpad and the same volume of SPG bufferwas injected into the left hind footpad. Footpad swelling was measuredat 48 H and 72 H following the injection. The difference between thethickness of the two footpads was used as a measure of the DTH response.Data are shown in FIG. 1 as the mean±SEM.

FIGS. 2A and 2B illustrate protection against MoPn infection with mompgene products following DNA immunization. Balb/c mice were immunizedwith (o) pcDNA3 (n=11), (●) pMOMP intramuscularly (n=12), (Δ) pMOMPintranasally (n=5) or (▴) MoPn EBs (n=12). Eighteen days after the lastimmunization, mice were challenged intranasally with infectious MoPn(1000 IFU). FIG. 2A shows body weight loss. Body weight was measureddaily following infection challenge and each point in FIG. 2A,represents the mean±SEM of the body weight loss. FIG. 2B shows in vivochlamydia clearance. Mice were sacrificed day 10 postinfection andrecovery of infectious MoPn from lung tissue was analyzed byquantitative tissue culture in order to determine the in vivo chlamydialclearance. The data in FIG. 2B, represent mean±SEM of the log₁₀ IFU perlung.

FIG. 3 illustrates detection of serum antibody to MoPn MOMP in DNAimmunized mice by immunoblot analysis. Day 60 pooled sera from miceimmunized with MoPn EBs (Lane A), pMOMP (Lane B), blank pcDNA3 vector(Lane C) or saline (Lane D), were diluted at 1:100 and reacted withpurified MoPn EBs that had been separated in a 10% SDS-polyacrylamidegel and transferred to a nitrocellulose membrane.

FIGS. 4A, 4B, 4C and 4D compare serum 1gG subclasses 1gG_(2a) (FIGS. 4Aand 4C) with lgG, FIGS. 4B and 4D) against recombinant MOMP protein(FIGS. 4A and 4B) or MoPn EBs (FIGS. 4C and 4D) induced by DNAimmunization. Mice were non-immunized or immunized intramuscularly withpMOMP, CTP synthetase DNA (pCTP) or the blank plasmid vector (pcDNA3) at0,3,6 weeks and pooled sera from each group were collected two weeksfollowing the last immunization (day 10). The data in FIGS. 4A and 4Brepresent mean±SEM of the OD value of four duplicates.

FIGS. 5A and 5B demonstrate that DNA vaccination with the MOMP geneenhanced clearance of MoPn infection in the lung. Groups of Balb/c micewere immunized with pMOMP (n=10), pcDNA3 (n=10) or saline (n=5).Eighteen days after the last immunization, the mice were challengedintranasally with infectious MoPn (10⁴IFU). FIG. 5A shows the bodyweight of the mice measured daily following challenge infection untilthe mice were sacrificed at day 10. Each point in FIG. 5A, representsthe mean±SEM of the body weight change. * represents P<0.05 comparedwith pcDNA3 treated group. FIG. 5B: the mice were sacrificed at day 10postinfection and the MoPn growth in the lung was analyzed byquantitative tissue culture. The data in FIG. 5B, represent mean±SEM ofthe Log₁₀IFU per lung. * represents P<0.01 compared with pcDNA3 treatedgroup.

FIGS. 6A and B, show evaluation of the responses of mice to MoPnintranasal challenge infection. FIG. 6A shows the change in body weightpost challenge and FIG. 6B shows the growth of MoPn in lung tissuecollected 10 days after challenge. Mice were sham immunized, immunizedintraperitoneally with MoPn EBs recovered from prior MoPn lunginfection, or immunized intramuscularly with p½MOMP. * represents P<10³¹³ compared to the pcDNA3 treated group. ** represents P<10⁻⁴ compared tothe pcDNA3 treated group.

FIG. 7 shows the elements and construction of plasmid pcDNA3/MOMP, 6495bp in size.

FIG. 8 shows schematically the nucleotide structure of the mature MOMPgene of C. trachomatis MoPn strain with conserved (CD) and variable (VD)domains identified as well as clones formed by cloning the identifiedsequences into pcDNA3, as described below in the Examples.

FIG. 9 shows the loss in body weight (in grams) following intranasalchallenge with 5×10³ IFU of MoPn among groups of Balb/c miceintramuscularly immunized with blank vector (pcDNA3), with pcDNA3 intowhich is individually cloned CV1 to CD5 encoding MOMP nucleotidesequences (CV1 etc), and with pcDNA3 into which the whole MOMP encodingnucleotide sequence is cloned (pMOMP).

FIG. 10 shows the results of assays to determine growth of C.trachomatis on day 10 in lungs of mice challenged with 5×10³ IFU of MoPnfollowing intramuscular immunization with blank vector (pcDNA3), withpcDNA3 into which is individually cloned CV1 to CD5 encoding MOMPnucleotide sequences (pCV1 etc), and with pcDNA3 into which the wholeMOMP encoding nucleotide sequence is cloned (pMOMP).

FIG. 11 shows footpad swelling reactions (DTH) 48 hours after footpadinjection of 2×10⁵ IFU of inactivated MoPn EBs among groups of Balb/cmice intramuscularly immunized with blank pcDNA3 vector (PC), withpcDNA3 into which is individually cloned CV1 to CD5 encoding MOMPnucleotide sequences (CV1 etc), and with pcDNA3 into which the wholeMOMP encoding nucleotide sequence is cloned (pM).

FIG. 12 shows the proliferation responses of splenocytes at day 60 postimmunization after in vitro stimulation with whole inactivated MoPn EBsfor 96 hours among groups of Balb/c mice immunized with blank pcDNA3vector (pc), with pcDNA3 into which is individually cloned CV1 to CD5encoding MOMP nucleotide sequences (CV1 etc), and with pcDNA3 into whichthe whole MOMP encoding nucleotide sequences is cloned (pM).

FIG. 13 shows the proliferation responses of splenocytes to the sameconstructs is in FIG. 11, except that the results are expressed as astimulation index (SI).

FIG. 14 shows the interferon-γ secretion response of MoPn stimulatedsplenocytes collected on day 60 after immunization among groups ofBalb/c mice immunized with blank pcDNA3 vector (pc), with pcDNA3 intowhich is individually cloned CV1 to CD5 encoding MOMP nucleotidesequences (CV1 etc), and with pcDNA3 into which the whole MoPn MOMPencoding nucleotide sequence is cloned (pM).

FIG. 15 shows the IgG2a antibody titer to whole MoPn EBs using seracollected at day 60 after immunization among groups of Balb/c miceimmunized with blank pcDNA3 vector (pc), with pcDNA3 into which isindividually cloned CV1 to CD5 encoding MOMP nucleotide sequences (CV1etc), and with pcDNA3 into which the whole MOMP encoding nucleotidesequences is cloned (pM).

FIG. 16 shows the IgG2a antibody titer to whole MoPn EBs using seracollected at day 60 after intramuscularly immunizing groups of Balb/cmice with blank pcDNA3 vector (pc), pcDNA3 containing the whole MoPnencoding nucleotide sequence (pM), and with pcDNA3 containing the wholeserovar C MOMP encoding nucleotide sequence (pM(C)).

FIG. 17 shows the 48 hour footpad swelling responses (DTH) to injectionwith 2×10⁵ IFU whole inactivated MoPn EBs among groups of Balb/c miceintramuscularly immunized 60 days previously with empty plasmid pcDNA3vector (pc), pcDNA3 containing the whole MoPn encoding nucleotidesequence (pM), and with pcDNA3 containing the whole serovar C MOMPencoding nucleotide sequence (pM(C)).

FIG. 18 shows the 96 hour proliferation of MoPn EB simulatedsplenocytes, expressed as a stimulation index (SI), collected fromgroups of Balb/c mice intramuscularly immunized with empty plasmidpcDNA3 vector (pc), pcDNA3 containing the whole MoPn MOMP encodingnucleotide sequence (pM), and with pcDNA3 containing the whole serovar Cencoding nucleotide sequence (pM(C)) sixty days previously.

FIG. 19 shows the IFN-γ secretion of MoPn EBs stimulated splenocytescollected from groups of Balb/c mice intramuscularly immunized 60 dayspreviously with empty pcDNA3 plasmid (pc), pcDNA3 containing the wholeMoPn MOMP encoding nucleotide sequence (pM), and with pcDNA3 containingthe whole serovar C encoding nucleotide sequence (pM(C)).

FIGS. 20A to 20F show a comparison of the amino acid sequence of MOMPsequences (SEQ ID NOS: 1 to 15) from a variety of serovars of C.trachomatis. Residues which are identical to serovar E MOMP arerepresented by dots. The four VDs (VDI to VDIV) and the conservedcysteines are boxed by solid line. The conserved position where onecysteine is located in all C. trachomatis and C. pneumonitis MOMPsequences, but where one serine is located in GPIC and Mn MOMPs, isboxed by a broken line. Numbers above boxes denote amino acid residuesof serovar E MOMP only.

GENERAL DESCRIPTION OF THE INVENTION

To illustrate the present invention, plasmid DNA was constructedcontaining the MOMP gene and MOMP gene fragments from the C. trachomatismouse pneumonitis strain (MoPn), which is a natural murine pathogen,permitting experimentation to be effected in mice. It is known thatprimary infection in the model induces strong protective immunity toreinfection. For human immunization, a human pathogen strain is used,such as serovar C of C. trachomatis.

Any convenient plasmid vector may be used for the MOMP gene or fragment,such as pcDNA3, a eukaryotic II-selectable expression vector(Invitrogen, San Diego, Calif., USA), containing a cytomegaloviruspromoter. The MOMP gene or MOMP gene fragment may be inserted in thevector in any convenient manner. The gene or gene fragments may beamplified from Chlamydia trachomaticgenomic DNA by PCR using suitableprimers and the PCR product cloned into the vector. The MOMPgene-carrying plasmid may be transferred, such as by electroporation,into E. coli for replication therein. A MOMP-carrying plasmid,pcDNA3/MOMP, of 6495 bp in size, is shown in FIG. 7. Plasmids may beextracted from the E. coli in any convenient manner.

The plasmid containing the MOMP gene or MOMP gene fragment may beadministered in any convenient manner to the host, such asintramuscularly or intranasally, in conjunction with apharmaceutically-acceptable carrier. In the experimentation outlinedbelow, it was found that intranasal administration of the plasmid DNAelicited the strongest immune response.

The data presented herein and described in detail below demonstratesthat DNA immunization with the C. trachomatis MOMP gene and MOMP genefragments elicits both cellular and humoral immune responses andproduces significant protective immunity to lung challenge infectionwith C. trachomatis MoPn. The results are more encouraging than thoseobtained using recombinant MOMP protein or synthetic peptides as theimmunogen and suggest that DNA immunization is an alternative method todeliver a chlamydial subunit immunogen in order to elicit the requisiteprotective cellular and humoral immune responses.

The data presented herein also demonstrate the importance of selectionof an antigen gene or gene fragment for DNA immunization. The antigengene elicits immune responses that are capable of stimulating recallimmunity following exposure to the natural pathogen. In particular,injection of a DNA expression vector encoding the major outer surfaceprotein (pMOMP) or fragment thereof but not one encoding a cytoplasmicenzyme (CTP synthetase) of C. trachomatis, generated significantprotective immunity to subsequent chlamydial challenge. The protectiveimmune response appeared to be predominantly mediated by cellularimmunity and not by humoral immunity since antibodies elicited by DNAvaccination did not bind to native EBs. In addition, MOMP DNA but notCTP synthetase DNA immunization elicited cellular immunity readilyrecalled by native EBs as shown by positive DTH reactions.

In addition, mucosal delivery of MOMP DNA is demonstrated herein to besignificantly more efficient in inducing protective immunity to C.trachomatis infection than intramuscular injection. This may be relevantto the nature of C. trachomatis infection which is essentiallyrestricted to mucosal surfaces and the efficiency of antigenpresentation (ref. 14). The rich population and rapid recruitment ofdendritic cells into the respiratory epithelium of the lung may berelevant to the enhanced efficacy of intranasal DNA immunizationexperiments (ref. 15). The data presented herein represents thedemonstration of a first subunit chlamydial vaccine which engenderssubstantial protective immunity.

Additionally, it may be possible to amplify (and/or canalize) theprotective immune response by co-administration of DNAs that expressimmunoregulatory cytokines in addition to the antigen gene in order toachieve complete immunity (ref. 21) The use of multiple antigen genesfrom chlamydia may augment the level of protective immunity achieved byDNA vaccination.

A possible concern regarding MOMP DNA immunization stems from theobservation that the MOMP among human C. trachomatis strains is highlypolymorphic (ref. 16) and hence it may be difficult to generate auniversal chlamydial vaccine based on this antigen gene. One way tosolve this problem may be to search for conserved protective epitope(s)within the MOMP molecule. As seen in the results presented below,certain vectors containing nucleotide sequences encoding conserved andvariable domains, identified in FIG. 8, or conserved domains generated aprotective immune response, as determined by loss of body weight, asshown in FIG. 9. FIG. 10 shows that the pCV3 and pCD5 immunogen evoked aprotective immune response to MoPn challenge as measured by in vivogrowth of MoPn in lung tissue day 10, with challenge and comparable topMOMP.

FIGS. 12 and 13 show the proliferation responses of splenocytes to thevectors containing the conserved and variable domains and the whole MOMPgene. These responses were determined in the following manner. Mice weresacrificed two weeks after the fourth immunization. The spleens wereremoved and single-cell suspensions were prepared. 200 μl of the cellsuspension (5×10⁵ well) in RPMI-1640 medium containing 10%heat-inactivated fetal calf serum (FCS), 1% L-glutamine and 5×10⁻⁵ M2-mercaptoethanol (2ME, Kodak, Rochester, N.Y.) were incubated with1×10⁵ IFU/ml of MoPn in 96 well flat bottom plates in triplicate 37° C.in 5% CO₂ for 96 hours. Negative control wells contained spleen cellswithout antigen and positive control wells contained spleen cells with0.25 μg/ml of concanavalin A. 0.25 μCi/well of tritiated (³H) thymidine(2 Ci/mmol, 74 Gbq/mmol, imCi/ml, ICN, Irvine, Calif.) was added after 3days of culture and 16 h before harvest. The cells were harvested with aPHD cell harvester (Cambridge Technology Inc., Watertown, Mass., USA)and counted in 2 ml of scintillation solution (Universal, ICN, CostaMesa) in a Beckman LS5000 counter (Beckman Instrument, UK).

The results obtained are set forth in FIGS. 12 and 13, which show thatpCV3 and pMOMP elicit a cell mediated immune response.

FIG. 14, which shows interferon-γ secretion responses of thesplenocytes, to the vectors containing the conserved and variabledomains and the whole MOMP gene. These responses were determined in thefollowing manner. A cytokine-specific ELISPOT assay was used for thequantification of murine. IFNγ and IL-10 secreting cells in the murinespleen. For all assays 96-well nitrocellulose-based microtiters(Milititer Multiscreen HA plates, Millipore Corp, Molshem, France) werecoated overnight at 4° C. with 100 μl of the anti-cytokine mAb dilutedin PBS at a concentration of 5 μg/ml. After removing the coatingsolution from the plates, wells were blocked for at least 1 hour withRPMI-1640 media containing 40% fetal calf serum at 37° C., in CO²⁻.After rinsing the plates with PBS-T once, the testing cells were addedinto the wells.

For induction of antigen specific IFNγ secreting cells in immunizedmice, single cells were adjusted to 5×10⁶ cells/ml and cultured with2×10⁵ IFU/ml of UV-killed EB of MoPn in 24 well plates for 72 hours.After washing with RPMI 1640, cells were added onto the 96-well platesfor 72 hours. After washing with RPMI 1640, cells were added onto the96-well nitrocellulose-based microtiter plates which had been previouslycoated with anti-cytokine antibodies. The cells were added to individualwells (2×10⁵ or 1×10⁵/100-μl/well) and incubated for 24 hours at 37° C.in a CO₂ incubator. Wells were rinsed extensively with PBS-T containing1% BSA. Following rinsing with PBS-T three times (removing thesupporting manifold and washing the back of the plate thoroughly withPBS-T), alkaline phosphatase conjugated streptavidin in PBS containing1% BSA at 1:2000 at a concentration of 0.5 μg/ml was added and incubatedat 37° C. in CO₂ for 45 min. After rinsing thoroughly, 100 μl/well ofthe colormetric substrate phosphate BICP (5-bromo-4-chloro-3-indolylphosphate)/NBT (Nitro blue tetrazolium) at 0.16 mg/ml BICP and 1 mg/mlNBT in substrate buffer (0.1 M NaCl, 0.1M Tris, pH 9.5, 0.05 M MgCl₂)was added and incubated at room temperature until spots were visualized.The reaction was stopped by the addition of water.

The results obtained in FIG. 14 suggest that cytokine generation may notnecessarily be a correlate of a protective immune response.

FIG. 15 shows IgG_(2a) antibody titers in sera collected from the mice60 days immunization by the vectors containing the conserved andvariable domains and full length MOMP gene. Only in the case ofimmunization by pCV3 and pCV5, was an IgG_(2a) immune responsegenerated, indicating that a Th1-like response was elicited by thesevectors.

Another, possibly more feasible, way is to design a multivalent vaccinebased on multiple MOMP genes. The latter approach is justified by thefact that the inferred amino acid sequences of MOMP among relatedserovars is relatively conserved (see FIGS. 20A to 20F) and therepertoire of C. trachomatis gene variants appears to be finite (ref.16). As may be seen from the data presented in the Examples below, apartially non-reactive immune response was elicited by the MOMP gene ofserovar C of C. trachomatis to the MOMP gene of serovar MoPn of C.trachomatis (FIGS. 16 to 19).

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis and treatment of chlamydial infections.A further non-limiting discussion of such uses is further presentedbelow.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from the MOMP genes or fragments thereof and vectors asdisclosed herein. The vaccine elicits an immune response in a subjectwhich includes the production of anti-MOMP antibodies. Immunogeniccompositions, including vaccines, containing the nucleic acid may beprepared as injectables, in physiologically-acceptable liquid solutionsor emulsions for polynucleotide administration. The nucleic acid may beassociated with liposomes, such as lecithin liposomes or other liposomesknown in the art, as a nucleic acid liposome (for example, as describedin WO 9324640) or the nucleic acid may be associated with an adjuvant,as described in more detail below. Liposomes comprising cationic lipidsinteract spontaneously and rapidly with polyanions, such as DNA and RNA,resulting in liposome/nucleic acid complexes that capture up to 100% ofthe polynucleotide. In addition, the polycationic complexes fuse withcell membranes, resulting in an intracellular delivery of polynucleotidethat bypasses the degradative enzymes of the lysosomal compartment.Published PCT application WO 94/27435 describes compositions for geneticimmunization comprising cationic lipids and polynucleotides. Agentswhich assist in the cellular uptake of nucleic acid, such as calciumions, viral proteins and other transfection facilitating agents, mayadvantageously be used.

Polynucleotide immunogenic preparations may also be formulated asmicrocapsules, including biodegradable time-release particles. Thus,U.S. Pat. No. 5,151,264 describes a particulate carrier of aphospholipid/glycolipid/polysaccharide nature that has been termed BioVecteurs Supra Moléculaires (BVSM). The particulate carriers areintended to transport a variety of molecules having biological activityin one of the layers thereof.

U.S. Pat. No. 5,075,109 describes encapsulation of the antigenstrinitrophenylated keyhole limpet hemocyanin and staphylococcalenterotoxin B in 50:50 poly(DL-lactideco-glycolide). Other polymers forencapsulation are suggested, such as poly(glycolide),poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters andpoly(8-hydroxybutyric acid), and polyanhydrides.

Published PCT application WO 91/06282 describes a delivery vehiclecomprising a plurality of bioadhesive microspheres and antigens. Themicrospheres being of starch, gelatin, dextran, collagen or albumin.This delivery vehicle is particularly intended for the uptake of vaccineacross the nasal mucosa. The delivery vehicle may additionally containan absorption enhancer.

The MOMP gene containing non-replicating vectors may be mixed withpharmaceutically acceptable excipients which are compatible therewith.Such excipients may include, water, saline, dextrose, glycerol, ethanol,and combinations thereof. The immunogenic compositions and vaccines mayfurther contain auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessthereof. Immunogenic compositions and vaccines may be administeredparenterally, by injection subcutaneously, intravenously, intradermallyor intramuscularly, possibly following pretreatment of the injectionsite with a local anesthetic. Alternatively, the immunogeniccompositions formed according to the present invention, may beformulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkylene glycols ortriglycerides. Oral formulations may include normally employedincipients, such as, for example, pharmaceutical grades of saccharine,cellulose and magnesium carbonate.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizethe MOMP and antibodies thereto, and if needed, to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgement of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of about 1 μg to about 1 mg of theMOMP gene-containing vectors. Suitable regimes for initialadministration and booster doses are also variable, but may include aninitial administration followed by subsequent administrations. Thedosage may also depend on the route of administration and will varyaccording to the size of the host. A vaccine which protects against onlyone pathogen is a monovalent vaccine. Vaccines which contain antigenicmaterial of several pathogens are combined vaccines and also belong tothe present invention. Such combined vaccines contain, for example,material from various pathogens or from various strains of the samepathogen, or from combinations of various pathogens.

Immunogenicity can be significantly improved if the vectors areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Thus,adjuvants have been identified that enhance the immune response toantigens. Some of these adjuvants are toxic, however, and can causeundesirable side-effects, making them unsuitable for use in humans andmany animals. Indeed, only aluminum hydroxide and aluminum phosphate(collectively commonly referred to as alum) are routinely used asadjuvants in human and veterinary vaccines.

A wide range of extrinsic adjuvants and other immunomodulating materialcan provoke potent immune responses to antigens. These include saponinscomplexed to membrane protein antigens to produce immune stimulatingcomplexes (ISCOMS), pluronic polymers with mineral oil, killedmycobacteria in mineral oil, Freund's complete adjuvant bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as Quil A derivatives and components thereof, QS 21, calciumphosphate, calcium hydroxide, zinc hydroxide, an octodecyl ester of anamino acid, ISCOPREP, DC-chol. DDBA and polyphosphazene. Advantageouscombinations of adjuvants are described in U.S. patent applications Ser.Nos.: 08/261,194 filed Jun. 16, 1994 (now U.S. Pat. No. 6,764,682) andSer No. 08/483.856 filed Jun. 7, 1995 (now U.S. Pat. No. 5,837,250),assigned to the assignee hereof and the disclosures of which areincorporated herein by reference thereto (WO 95/34308).

In particular embodiments of the present invention, the non-replicatingvector comprising a first nucleotide sequence encoding a MOMP gene ofChlamydia may be delivered in conjunction with a targeting molecule totarget the vector to selected cells including cells of the immunesystem.

The non-replicating vector may be delivered to the host by a variety ofprocedures, for example, Tang et al. (ref. 17) disclosed thatintroduction of gold microprojectiles coated with DNA encoding bovinegrowth hormone (BGH) into the skin of mice resulted in production ofanti-BGH antibodies in the mice, while Furth et al. (ref. 18) showedthat a jet injector could be used to transfect skin, muscle, fat andmammary tissues of living animals.

2. Immunoassays

The MOMP genes, MOMP gene fragments and vectors of the present inventionalso are useful as immunogens for the generation of anti-MOMP antibodiesfor use in immunoassays, including enzyme-linked immunosorbent assays(ELISA), RIAs and other non-enzyme linked antibody binding assays orprocedures known in the art. In ELISA assays, the non-replicating vectorfirst is administered to a host to generate antibodies specific to theMOMP. These MOMP specific antibodies are immobilized onto a selectedsurface, for example, a surface capable of binding the antibodies, suchas the wells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed antibodies, a nonspecific protein, such as asolution of bovine serum albumin (BSA) that is known to be antigenicallyneutral with regard to the test sample, may be bound to the selectedsurface. This allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This procedure may includediluting the sample with diluents, such as solutions of BSA, bovinegamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. Thesample is then allowed to incubate for from about 2 to 4 hours, attemperatures such as of the order of about 20° to 37° C. Followingincubation, the sample-contacted surface is washed to removenon-immunocomplexed material. The washing procedure may include washingwith a solution, such as PBS/Tween or a borate buffer. Followingformation of specific immunocomplexes between the test sample and thebound MOMP specific antibodies, and subsequent washing, the occurrence,and even amount, of immunocomplex formation may be determined.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

Example 1

This Example illustrates the preparation of a plasmid vector containingthe MOMP gene.

pMOMP expression vector was made as follows. The MOMP gene was amplifiedfrom Chlamydia trachomatis mouse pneumonitis (MoPn) strain genomic DNAby polymerase chain reaction (PCR) with a 5′ primer(GGGGATCCGCCACCATGCTGCCTGTGGGGAATCCT) (SEQ ID NO: 16) which includes aBamH1 site, a ribosomal binding site, an initiation codon and theN-terminal sequence of the mature MOMP of MoPn and a 3′ primer(GGGGCTCGAGCTATTAACGGAACTGAGC) (SEQ ID NO: 17) which includes theC-terminal sequence of the MoPn MOMP, a Xhol site and a stop codon. TheDNA sequence of the MOMP leader peptide gene sequence was excluded.After digestion with BamH1 and Xhol, the PCR product was cloned into thepcDNA3 eukaryotic II-selectable expression vector (Invitrogen, SanDiego) with transcription under control of the human cytomegalovirusmajor intermediate early enhancer region (CMV promoter). The MOMPgene-encoding plasmid was transferred by electroporation into E. coliDH5αF which was grown in LB broth containing 100 μg/ml of ampicillin.The plasmids was extracted by Wizard™ Plus Maxiprep DNA purificationsystem (Promega, Madison). The sequence of the recombinant MOMP gene wasverified by PCR direct sequence analysis, as described (ref. 20).Purified plasmid DNA was dissolved in saline at a concentration of 1mg/ml. The DNA concentration was determined by a DU-62 spectrophotometer(Beckman, Fullerton, Calif.) at 260 nm and the size of the plasmid wascompared with DNA standards in ethidium bromide-stained agarose gel.

The MOMP gene containing so obtained plasmid, pcDNA3/MOMP, and itsconstitutive elements are shown in FIG. 7. A similar plasmid (pM(C)) wasconstructed from the MOMP gene serovar C of C. trachomatis.

Example 2

This Example illustrates DNA immunization of mice and the results of DTHtesting.

A model of murine pneumonia induced by the C. trachomatis mousepneumonitis strain (MoPn) was used (ref. 11). Unlike most strains of C.trachomatis which are restricted to producing infection and disease inhumans, MoPn is a natural murine pathogen. It has previously beendemonstrated that primary infection in this model induces strongprotective immunity to reinfection. In addition, clearance of infectionis related to CD4 Th1 lymphocyte responses and is dependent on MHC classII antigen presentation (ref. 11).

For experimental design, groups of 4 to 5 week old female Balb/c mice (5to 13 per group) were immunized intramuscularly (IM) or intranasally(IN) with plasmid DNA containing the coding sequence of the MoPn MOMPgene (1095 bp), prepared as described in Example 1, or with the codingsequence of the C. trachomatis serovar L₂ CTP synthetase gene (1619 bp(refs. 10, 12), prepared by a procedure analogous described inExample 1. CTP synthetase is a conserved chlamydial cytoplasmic enzymecatalizing the final step in pyrimidine biosynthesis and is not known toinduce protective immunity. Negative control animals were injected withsaline or with the plasmid vector lacking an inserted chlamydial gene.

For IM immunization, both quadriceps were injected with 100 μg DNA in100 μl of saline per injection site on three occasions at 0, 3 and 6weeks. For IN immunization anaesthetized mice aspirated 25 μd of salinecontaining. 50 μg DNA on three occasions at 0, 3 and 6 weeks. As apositive control, a separate group of mice received 5×10⁶ inclusionforming units (IFUs) of MoPn EBs administered intraperitoneally inincomplete Freund's adjuvant according to the above schedule. At week 8,all groups of mica had sera collected for measuring antibodies and weretested for delayed-type hypersensitivity (DTH) to MoPn Ebs by footpadinjection (ref. 13).

A positive 48 and 72 hour DTH reaction was detected among mice immunizedwith MOMP DNA or with MoPn Ebs but not among mice immunized with theblank vector (see FIG. 1). The DTH reaction elicited with MOMP DNAdelivered intranasally was comparable to that observed among miceimmunized with EBs. No DTH reaction was detected among the groups ofmice vaccinated with CTP synthetase DNA (see Table 1 below). Thus,injection of MOMP DNA generated a DTH reaction that was capable ofrecall by naturally processed peptides from C. trachomatis EBs whileinjection of CTP synthetase DNA failed to do so.

Example 3

This Example illustrates DNA immunization of mice and the generation ofantibodies.

Injection of CTP synthetase DNA as described in Example 2 resulted inthe production of serum antibodies to recombinant CTP synthetase(Table 1) (ref. 14). Antigen-specific serum Abs were measured by ELISA.Flat-bottom 96-well plates (Corning 25805, Corning Science Products,Corning, N.Y.) were coated with either recombinant chlamydialCTP-synthetase (1 μg/ml) or purified MoPn EBs (6×10⁴ IFU/well) overnightat 4° C. The Plates were rinsed with distilled water and blocked with 4%BSA PBS-Tween and 1% low fat skim milk for 2 hours at room temperature.Dilutions of sera samples were performed in 96-well round bottom platesimmediately prior to application on the antigen coated plates. Theplates were incubated overnight at 4° C. and washed ten times.Biotinylated goat anti-mouse IgG1 or goat anti-mouse IgG2a (SouthernBiotechnology Associates, Inc. Birmingham, Ala.) were next applied for 1hour at 37° C. After washing; streptoavidin-alkaline phosphataseconjugate (Jackson ImmunoResearch Laboratories, Inc. Mississagua,Ontario, Canada) were added and incubated at 37° C. for 30 min.Following another wash step, phosphatase substrate in phosphatase buffer(pH 9.8) was added and allowed to develop for 1 hour. The plates wereread at 405 nm on a BIORAD 3550 microplate reader.

IgG2a antibody titers were approximately 10-fold higher thanIgG1antibody titers suggesting that DNA immunization elicited a moredominant T_(H1)-like response. Injection of MOMP DNA as described inExample 2 resulted in the production of serum antibodies to MOMP (Table2) as detected in an immunoblot assay (FIGS. 2A and 2B). However,neither CTP synthetase DNA nor MOMP DNA immunized mice producedantibodies that bound to native C. trachomatis EBs (Table 1), suggestingthat the antibody responses may not to be the dominantly protectivemechanism. A comparison of serum IgG subclasses, IgG2a (FIGS. 4A and 4Cand IgG₁ (FIGS. 4B and 4D) against MOMP protein (FIGS. 4A and 4B) orMoPn (FIGS. 4C and 4D) induced by DNA immunization as described above,is contained in FIGS. 4A to 4D.

Example 4

This Example illustrates DNA immunization of mice to achieve protection.

To investigate whether a cell-mediated immune response elicited by MOMPDNA was functionally significant, in vivo protective efficacy wasevaluated in mice challenged intranasally with 1×10³ IFU of C.trachomatis MoPn. To provide a measure of Chlamydia-induced morbidity,the loss in body weight was measured over 10 days following challengewith C. trachomatis (see FIG. 2A). Mice injected with the unmodifiedvector were used as negative controls and mice immunized with EBs wereused as positive controls. Mice immunized with MOMP DNA intranasallymaintained a body weight comparable to that observed among EB immunizedmice. Mice intramuscularly immunized with MOMP DNA lost body mass butdid so at a rate less than the negative control group.

A more direct measure of the effectiveness of DNA vaccination is theability of mice immunized with MOMP DNA to limit the in vivo growth ofChlamydiafollowing a sublethal lung infection. Day 10 post-challenge isthe time of peak growth (ref. 13) and was chosen for comparison of lungtiters among the various groups of mice. Mice intranasally immunizedwith MOMP DNA had chlamydial lung titers that were over 1000-fold lower(log₁₀ IFU 1.3±0.3; mean±SEM) than those of control mice immunized withthe blank vector (log₁₀ IFU 5.0±0.3; p<0.01) (see FIG. 2B). Miceintramuscularly immunized with MOMP DNA had chlamydial lung titers thatwere more than 10-fold lower than the unmodified vector group (p=0.01).Mice intranasally immunized with MOMP DNA had significantly lowerchlamydial lung titers than mice immunized with MOMP DNA intramuscularly(log₁₀ IFU 1.3±0.8 versus log₁₀ IFU 0.66±0.3 respectively; p=0.38). Thesubstantial difference (2.4 logs) in chlamydial lung titers observedbetween the intranasally and intramuscularly MOMP DNA immunized micesuggests that mucosal immunization is more efficient at inducing immuneresponses to accelerate chlamydial clearance in the lung. The lack ofprotective effect with the unmodified vector control confirms that DNAper se was not responsible for the immune response. Moreover, theabsence of protective immunity following immunization with CTPsynthetase DNA confirms that the immunity was specific to the MOMP DNA(see Table 1). FIGS. 5A and 5B shows similar challenge data at a higherchallenge dose.

Example 5

This Example describes the construction of p½MOMP.

A PCR cloned MoPn gene was constructed containing a deletion mutation incodon 177. This mutation yields a truncated MOMP protein containingapproximately 183 amino-terminal amino acids (ref. 10). This construct,termed p½MOMP, was cloned into the vector pcDNA3 (Invitrogen), in themanner described in Example 1 for the full length MOMP gene.

In addition, a series of vectors was generated containing fragments ofthe nucleotide sequence of the MoPn MOMP gene by PCR cloning andsubsequent cloning into the vector pcDNA3 to generate plasmids pCV1,pCV2, pCV3, pCV4 and pCV5, respectively containing the portions of theMoPn MOMP gene shown in FIG. 8.

Example 6

This Example illustrates immunization of mice with p½MOMP, pCV1, pCV2,pCV3, pCV4 and pCV5.

Balb/c mice were immunized in the quadriceps three times at three weekintervals with 100 μg of p½MOMP, pCV1, pCV2, pCV3, pCV4 and pCV5 DNA.

Fifteen days after the last immunization and 60 days after the firstinjection, mice were bled for measurement of serum antibodies of MoPnEBs in an EIA assay and were injected in the footpad with 25 μl (5×10⁴inclusion forming units) of heat killed EBs for measurement of DTH whichwas measured at 72 hours (ref. 13). Mice were intranasally challengedwith 1000 infectious units of MoPn and their body weight measured dailyfor the subsequent 10 days. At that time, mice were sacrificed andquantitative cultures of MoPn in the lung determined (ref. 13).

Table 3 shows that p½MOMP immunization elicited a positive DTH responseto footpad injection of MoPn EBs. Low titers (approximate titer 1/0.100)serum antibodies to surface determinants on EBs were also detected atday 60 post vaccination. Immunization with the unmodified vectorelicited neither serum antibodies nor a DTH response. FIG. 11 shows thatimmunization with pCV1, pCV2, pCV3, pCV4 and pCV5 elicited variablepositive DTH responses to footpad injection of MoPn EBs. pCV3 and pCD5elicited greater responses comparable to pMOMP.

FIG. 6A shows that p½MOMP immunization evoked a protective immuneresponse to MoPn challenge as measured by change in body weight postinfection and by the in vivo growth of MoPn in lung tissue day 10 postchallenge. The in vivo growth among saline treated mice waslog₁₀5.8±0.21 and among p½MOMP immunized mice was log₁₀3.9±0.25,p<0.001, FIG. 6B. As a positive control, mice immunized with heat killedMoPn EBs or recovered from prior infection with MoPn were markedly andequivalently protected against challenged infection (p<0.0001).

FIG. 9 shows that pCV2, pCV3 and pCD5 immunization evoked a protectiveimmune response to MoPn challenge as measured by loss in body weightpost infection comparable to that in mice protected against disease, asseen by lung titres. However, the specific domains eliciting theseimmune responses do not include those predicted in the art to containT-cell epitopes. In this regard, several groups have attempted to defineMOMP T-cell epitopes (refs. 22 to 26). All of those studies usedoverlapping synthetic peptides to various regions of the MOMP protein toprime mice. None of the predicted epitopes fall within regions that havebeen found to be protective.

As may be seen in this Example, using a frame-shift deletion mutant atcodon 177 of the MOMP gene, significant protective immunity to challengeinfection was elicited suggesting that protective sites can be found inthe amino terminal half of the protein. In addition, it has furthershown in this Example that the vectors containing specific segments ofthe MOMP gene were able to protect against disease, based on body weightloss, namely pCV2 and pCD5. In addition, vectors pCV3 and pCD5 were ableto protect against infection, based on lung titres.

Example 7

This Example illustrates the effect of DNA immunization of mice withpM(C).

The pcDNA3 vector containing the MOMP gene for serovar C of C.trachomatis, prepared as described in Example 1, was immunized into micefollowing the procedure of Example 2 and various results chartedgraphically in comparison to the results obtained using pMOMP from MoPnstrain.

In this regard, Ig2a antibody responses (FIG. 16), footpad swellingresponses (FIG. 17), proliferation of splenocytes (FIG. 18) and IFN-γsecretion (FIG. 19) were determined following the procedures of Example3, Example 2 and Example 6 respectively.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a methodof nucleic acid, including DNA, immunization of a host, includinghumans, against disease caused by infection by a strain of Chlamydia,specifically C. trachomatis, employing a non-replicating vector,specifically a plasmid vector, containing a nucleotide sequence encodinga major outer membrane protein (MOMP) of a strain of Chlamydia or afragment of MOMP which generates a MOMP-specific immune response and apromoter to effect expression of MOMP in the host. Modifications arepossible within the scope of this invention.

TABLE 1 Serum antibody titers and delayed-type hypersensitivity (DTH)responses and in vivo growth of Chlamydia trachomatis following pCTPsynthetase or MoPn EB immunization. Results are presented as means ±SEM. Anti-MoPn EB anti-rCTP synthetase log₁₀ IFU/lung antibodies (log₁₀)antibodies (log₁₀) Anti-EB DTH d10 post IgG1 IgG2a IgG1 IgG2a (mm × 10²)challenge Saline (n = 9) <2 <2 <2 <2 4.5 ± 1.5 4.9 ± 2.4 pCTP synthetase<2 <2 3.8 ± .3 4.7 ± .1 1.4 ± 1.5 4.7 ± .13 (n = 11) EB (n = 4) 5.0 ± .34.8 ± .3 3.6 ± .8 2.9 ± 0  15.2 ± 2.0  0

TABLE 2 Serum antibody Elisa titers to Chlamydia trachomatis mousepneumonitis recombinant MOMP and EBs were measured 60 days after theinitial immunization among mice immunized with blank vector alone(pcDNA3), vector containing the MOMP gene (pMOMP) and vector containingthe CTP synthetase gene (pCTP). Non-immunized mice were also tested.rMOMP EB Immunogen IgG2a IgG1 IgG2a IgG1 pcDNA3  <2.6* <2.6 <2.6 <2.6pMOMP 3.77 ± 0.1 2.90 ± 0.14 3.35 ± 0.11 <2.6 pCTP ND ND <2.6 <2.6Preimmunization <2.6 <2.6 <2.6 <2.6 *log₁₀ mean ± SE IgG isotype speciicantibody titer ND = not done

TABLE 3 Immune responses at day 60 following p½MOMP, EB or blank vector(pcDNA3) immunization of mice. EB IgG_(2a) DTH response antibody titerto EB Immunogen (log₁₀) (mm × 10²) EB (n = 13) 5.6 ± 0.4 9.6 ± 2.0p½MOMP (n = 13) 2.0 ± 0     6 ± 1.6 pcDNA3 (n = 13) 1.3 ± 0   1 ± 1

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1. A method of stimulating a recall immune response to Chlamydiatrachomatis, which comprises administering to said host an effectiveamount of a non-replicating vector comprising: a nucleotide sequenceencoding a major outer membrane protein (MOMP) of a strain of Chlamydiatrachomatis or a fragment of said MOMP that generates a MOMP-specificimmune response, and a promoter sequence operatively coupled to saidnucleotide sequence for expression of said MOMP or said MOMP fragment inthe host.
 2. The method of claim 1 wherein said nucleotide sequenceencodes a full-length MOMP.
 3. The method of claim 1 wherein saidnucleotide sequence encodes an N-terminal fragment of the MOMP ofapproximately half the size of full-length MOMP.
 4. The method of claim1 wherein said nucleotide sequence encodes a region comprising at leastone of the conserved domains 2, 3 and 5 of a major outer membraneprotein of a strain of Chlamydia.
 5. The method of claim 4 wherein saidnucleotide sequence encoding the conserved domain 2 and/or 3 furtherincludes a nucleotide sequence encoding a variable domain of the majorouter membrane protein immediately downstream of said conserved domain.6. The method of claim 4 wherein said nucleotide sequence encodes theconserved domain 5 of a major outer membrane protein of a strain ofChlamydia.
 7. The method of claim 1 wherein said promoter sequence isthe cytomegalovirus promoter.
 8. The method of claim 1 wherein saidnon-replicating vector comprises plasmid pcDNA3 containing said promoterinto which said nucleotide sequence is inserted in operative relation tosaid promoter sequence.
 9. The method of claim 1 wherein said immuneresponse is predominantly a cellular immune response.
 10. The method ofclaim 1 wherein said non-replicating vector is administeredintranasally.
 11. The method of claim 1 wherein said host is a humanhost.
 12. A method of using a gene encoding a major outer membraneprotein (MOMP) of Chlamydia trachomatis or a fragment of said MOMP thatgenerates a MOMP-specific immune response, to produce a recall immuneresponse to Chlamydia trachomatis in a host, which comprises: isolatingsaid gene, operatively linking said gene to at least one controlsequence to produce a non-replicating vector, said control sequencedirecting expression of said MOMP or said MOMP fragment when introducedinto a host to produce an immune response to said MOMP or MOMP fragment,and introducing said vector into a host.
 13. The method of claim 12wherein said gene encoding MOMP encodes a full length MOMP.
 14. Themethod of claim 12 wherein said gene encoding MOMP encodes an N-terminalfragment of the MOMP of approximately half the size of full-length MOMP.15. The method of claim 12 wherein said nucleotide sequence encodes aregion comprising at least one of the conserved domains 2, 3 and 5 of amajor outer membrane protein of a strain of Chlamydia.
 16. The method ofclaim 15 wherein said nucleotide sequence encoding the conserved domain2 and/or 3 further includes a nucleotide sequence encoding a variabledomain of the major outer membrane protein immediately downstream ofsaid conserved domain.
 17. The method of claim 15 wherein saidnucleotide sequence encodes the conserved domain 5 of a major outermembrane protein of a strain of Chlamydia.
 18. The method of claim 12wherein said control sequence is the cytomegalovirus promoter.
 19. Themethod of claim 12 wherein said non-replicating vector comprises plasmidpcDNA3 containing said control sequence into which said gene encodingMOMP is inserted in operative relation to said control sequence.
 20. Themethod of claim 12 wherein said immune response is predominantly acellular Immune response.
 21. The method of claim 12 wherein said vectoris introduced into said host intranasally.
 22. The method of claim 12wherein said host is a human host.